Quantification of Enteric Viruses,Pathogen Indicators,and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10⁴ MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.Over the last decade, thousands of people in the United States have been infected with waterborne diseases, a large number of whom were hospitalized. Most of the waterborne disease outbreaks in the United States that occurred between 1991 and 2004 were related to microbial agents, i.e., viruses, bacteria, and parasites (5, 30). The majority of the outbreaks involved unidentified agents, and the Environmental Protection Agency (EPA) suspects that many of the outbreaks due to unidentified sources were caused by enteric viruses (46). Indeed, viruses have a high potential for groundwater pollution due to their small size and low die-off rates. The occurrence of enteric viruses in groundwater has been reported (1, 7, 12, 17). In the United States, approximately 5.6 million dry tons of biosolids are generated annually and 60% of the biosolids are applied to land (36).Several studies have reported the occurrence of enteric viruses in biosolids (18, 35, 47); however, information on the quantity and infectivity of enteric viruses in biosolids is still limited, and most studies focused solely on enteroviruses (41). Few studies have reported the levels of human adenoviruses (HAdV) in biosolids (6, 47), and no quantitative results have yet been reported on some of the emerging viruses, such as hepatitis A virus (HAV) and noroviruses (NV). Also, only one or two types of enteric viruses were quantified in the previous studies; therefore, it is hard to determine and compare the prevalence of different types of enteric virus in biosolids, since the samples and sample processing methods varied from study to study. A few studies focused on the viral infectivity of biosolids, and the results showed that infectious astrovirus and enteroviruses were still present in the treated biosolids (9, 18, 42). However, no results on the occurrence of adenoviruses in biosolids have been reported.PCR techniques have been used in most of the recent environmental virology studies. In comparing these techniques to cell culture, the main advantages of PCR methods for virus detection are fast results, less labor intensiveness, high specificity and sensitivity, and the capability of detecting difficult-to-culture or nonculturable viruses (for examples, human noroviruses and adenovirus 40/41). Quantitative real-time PCR (qPCR), which is considered the latest advancement in PCR technology, can provide both qualitative and quantitative results. However, PCR results may not reflect the infectivity of the samples since PCR only detects the genes of the pathogens; therefore, integrated cell culture-PCR (ICC-PCR) was developed to identify the specific infectious enteric viruses. ICC-PCR has been used to detect infectious enteric viruses in river water, tap water, beach water, and wastewater effluent samples (28, 29, 39, 50). However, Buffalo green monkey (BGM) cell culture, currently recommended by the EPA, has been compared with other cell lines, such as A549 and PLC/PRC/5 (28, 39), and the results showed that enteric viruses were propagated better with these cell lines than with BGM cells.The main objective of this work was to investigate the occurrence and the quantitative levels of the enteric viruses in class B mesophilic anaerobically digested (MAD) biosolid samples by molecular and cell culture methods. These results can be used for risk assessment at biosolid application sites. Also, enteric viruses have been suggested as fecal source tracking indicators (21, 32), and the levels of enteric viruses in biosolids reported in this study would be useful for the determination of which enteric virus is a better fecal source tracking indicator at biosolid application sites. MAD biosolids were chosen since they are the most common type of class B biosolid produced in the United States (47). Three dewatered biosolid samples were also included for comparison purposes. The levels of human adenovirus (HAdV), adenovirus type 40/41 (HAdV 40/41), enterovirus (EV), norovirus GI (NV GI) and NV GII, human polyomavirus (HPyV), and hepatitis A virus (HAV) were quantified by qPCR methods. BGM and A549 cell lines were used to quantify the infectious viruses in the biosolids, and the effectiveness of these two cell lines'' ability to propagate infectious viruses was compared. The occurrence of infectious EV and HAdV in biosolids was determined by ICC-PCR, and the serotypes of the infectious adenoviruses propagated on A549 were further determined. The levels of pathogen indicators and Salmonella enterica are also presented in this study.     

Male-specific coliphages as indicators of thermal inactivation of pathogens in biosolids

Male-specific (F+) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F+ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F+ DNA and F+ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F+ RNA phages. Separate experiments on individual isolates at 53 degrees C confirmed the apparent heat resistance of group I F+ RNA coliphages as well as the susceptibility of group III F+ RNA coliphages. Although few F+ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F+ RNA phages. Hence, F+ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F+ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.     

qPCR quantification and genetic characterization of Clostridium perfringens populations in biosolids composted for 2 years

Aim: The ability of Clostridium perfringens to survive for a long time in the environment makes it a suitable indicator of faecal pollution, but its use as a routine indicator organism in biosolids and composted biosolids has not yet been adopted. This study was performed to improve our understanding of C. perfringens persistence in composted biosolids by monitoring its presence and studying its genetic diversity. Methods and Results: A culture‐independent TaqMan qPCR assay targeting the cpn60 gene was adapted to enumerate C. perfringens in composted biosolid samples varying in age from 1 to 24 months. The pathogen was detected in all compost samples under study, but no correlation between composting time and number of cpn60 copies was observed. Rep‐PCR detected 14 different C. perfringens genotypes, all belonging to toxinotype A, which is the most common biotype found in human and animal gastrointestinal tracts. Conclusions: Composting did not significantly decrease the number of C. perfringens cells. High genetic diversity of C. perfringens isolates present in composted biosolids is reported for the first time. Significance and Impact of Study: This study evaluated tools for surveillance of composting processes, source tracking and risk assessment of composted biosolids.     

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.     

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses'' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.     

Dynamics of C, N and P in soil amended with biosolids from a pharmaceutical industry producing cephalosporines or third generation antibiotics: a laboratory study

Large parts of the central highlands of Mexico are heavily eroded and the success of a planned reforestation program will greatly improve when the organic matter and nutrient content of the soil increases prior to the planting of the trees. This study investigated how the application of biosolids from a pharmaceutical company producing cephalosporines or third generation antibiotics could be used as a soil amendment and affect dynamics of C, P and N in soil. A sandy clay loam soil was sampled, amended with 24 g of dry biosolids kg(-1) dry soil or approximately 32 x 10(3) kg ha(-1) for the 0-10 cm layer, and incubated aerobically while production of carbon dioxide (CO(2)), dynamics of ammonium (NH(4)(+)),nitrite (NO(2)(-)), nitrate (NO(3)(-)), sodium bicarbonate (NaHCO(3)) extractable phosphorus (PO(4)(3-)), and microbial biomass carbon (C) were monitored. Results showed that the biosolid with pH 12, organic C content 162 g kg(-1), total N 21 g kg(-1), was of excellent quality considering its heavy metal content (USEPA) and a class "B" (USEPA) biosolid considering the amount of pathogens. No cephalosporines could be detected in the biosolid. Addition of biosolid to soil increased production of CO(2) 1.4 times and added >60 mg NH(4)(+) kg(-1). The application of biosolids did not significantly increase the concentration of NO(2)(-) which remained <2 mg N kg(-1) soil, but the concentration of NO(3)(-) did increase with 175 mg N kg(-1) soil. The microbial biomass C did not change when sewage biosolids was added and concentrations of extractable PO(4)(3-) only increased temporarily. Washing the biosolids reduced concentrations of NH(4)(+) and NO(3)(-), but also reduced pathogens and concentrations of chloride (Cl(-)), which might pose a treat to humans and the environment, respectively. Although the biosolid added valuable nutrients to the soil and did not inhibit C and N mineralization, further investigation into possible long-term environmental effects on soil processes and plant growth is necessary before this biosolid can be used in the field.     

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

AIM: To compare the inactivation rates of male-specific bacteriophage-2 (MS2), hepatitis A HM-175/18f (HM-175) and reovirus T1 Lang (T1 L) during alkaline stabilization of wastewater residues. METHODS AND RESULTS: A bench scale alkaline stabilization model was used to evaluate the inactivation of MS2 seeded into raw sludge simultaneously with HM-175 or T1 L. Stabilization was performed in triplicate at 28 and 4 degrees C for both viral combinations. During stabilization at 28 and 4 degrees C, MS2 and T1 L concentrations were similar at each time point (t = 0.1, 2, 12 and 24 h). MS2 and HM-175 concentrations were also similar at each time point during stabilization at 28 degrees C. At 4 degrees C, MS2 and HM-175 concentrations were not similar at the first two time points (t = 0.1 and 2 h), but were similar at later time points (t = 12 and 24 h). CONCLUSIONS: The inactivation rates of T1 L at 4 degrees C and both T1 L and HM-175 at 28 degrees C were similar to the inactivation rate of MS2 at all time points. At 4 degrees C, MS2 was inactivated at a faster rate during the first two time points (t = 0.1 and 2 h) than HM-175, but was inactivated similarly at later time points (t = 12 and 24 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Phages, such as MS2, would be ideal indicators for the presence of enteric viruses in wastewater residues because of their ubiquity, nonpathogenic nature, low cost and time associated with their detection. The findings of this study suggest that MS2 could serve as an indicator for monitoring the persistence of enteric viruses, such as HM-175 and T1 L, during alkaline stabilization performed at moderate temperatures (28 degrees C), but may not serve as an indicator for HM-175 at reduced temperature (4 degrees C). The utility of MS2 as an indicator of viral persistence during biosolids treatment should be further evaluated, as the increased efficiency and frequency of pathogen monitoring associated with their use may reduce the potential public health risk associated with biosolids, facilitating a greater acceptance for their land application.     

Bench and full-scale studies for odor control from lime stabilized biosolids: the effect of mixing on odor generation

Lime stabilization is a means to raise the pH of biosolids to meet specific pathogen requirements. Along with controlling the microbial growth, lime stabilization reduces the potential for offensive odors. Lime stabilized biosolids can be beneficially used as a soil amendment and also for land reclamation. However, if biosolids are not properly incorporated with the lime, there is a potential for microbial growth, which consequently leads to the emanation of offensive odors and growth of pathogens. Proper mixing was found to be an important factor for the reduction of offensive odors in biosolids treatment. To better understand the effects of mixing on odorous products, bench-scale and full-scale tests were conducted to assess the lime stabilization process and investigate mixing quality at a wastewater treatment plant to help reduce odors associated with known odorants. The results of 4-week laboratory bench-scale tests showed that mixing had the largest effect on odor. The hedonic tone test of the control samples with poor mixing showed a hedonic tone of -2.9 initially and then dropped to -7.3 on Day 29. The hedonic tone of the 3.5%, 7%, and 10% lime mixed biosolids had similar hedonic tones (-2.8 to -2.5) on Day 1 and slightly fluctuated over time and ended at -1.6 to -2.7 on Day 29, which was less odorous than the controls. The control sample with poor mixing showed a rapid pH drop from 12.1 on Day 1 to 8.4 on Day 7. The pH of the control sample was considerably lower than the mixed samples and ended up on Day 28 with a pH of 8.0. The pH of the 7% and 10% samples were relatively stable throughout the 4-week period with a pH still higher than 12 on Day 28. The biosolids with better mixing had a less offensive odor and weaker odor strength than the controls collected at the plant with poor mixing. The lime stabilization process in the wastewater treatment plant was modified in a full-scale study by prolonging the mixing time. The samples collected from the modified process had a less offensive odor and weaker odor strength than the controls from the original lime addition process with short mixing time. The hedonic tone of the two biosolid samples taken from the modified process (-2.5 to -3.0) was less negative than the controls (-3.5 to -4.5). The pH of the biosolid samples taken from the modified process was still above 12 on Day 21, while the pH notably drooped in the controls from 11 on Day 1 to 7.5-8.5 on Day 7. In addition, headspace analysis from both bench and full-scale tests indicated that the sulfur compounds were more prominent in poorly mixed samples than well mixed samples.     

Male-Specific Coliphages as Indicators of Thermal Inactivation of Pathogens in Biosolids

Male-specific (F⁺) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F⁺ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F⁺ DNA and F⁺ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F⁺ RNA phages. Separate experiments on individual isolates at 53°C confirmed the apparent heat resistance of group I F⁺ RNA coliphages as well as the susceptibility of group III F⁺ RNA coliphages. Although few F⁺ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F⁺ RNA phages. Hence, F⁺ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F⁺ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.     

Source tracking aerosols released from land-applied class B biosolids during high-wind events

DNA-based microbial source tracking (MST) methods were developed and used to specifically and sensitively track the unintended aerosolization of land-applied, anaerobically digested sewage sludge (biosolids) during high-wind events. Culture and phylogenetic analyses of bulk biosolids provided a basis for the development of three different MST methods. They included (i) culture- and 16S rRNA gene-based identification of Clostridium bifermentans, (ii) direct PCR amplification and sequencing of the 16S rRNA gene for an uncultured bacterium of the class Chloroflexi that is commonly present in anaerobically digested biosolids, and (iii) direct PCR amplification of a 16S rRNA gene of the phylum Euryarchaeota coupled with terminal restriction fragment length polymorphism to distinguish terminal fragments that are unique to biosolid-specific microorganisms. Each method was first validated with a broad group of bulk biosolids and soil samples to confirm the target's exclusive presence in biosolids and absence in soils. Positive responses were observed in 100% of bulk biosolid samples and in less than 11% of the bulk soils tested. Next, a sampling campaign was conducted in which all three methods were applied to aerosol samples taken upwind and downwind of fields that had recently been land applied with biosolids. When average wind speeds were greater than 5 m/s, source tracking results confirmed the presence of biosolids in 56% of the downwind samples versus 3% of the upwind samples. During these high-wind events, the biosolid concentration in downwind aerosols was between 0.1 and 2 microg/m3. The application of DNA-based source tracking to aerosol samples has confirmed that wind is a possible mechanism for the aerosolization and off-site transport of land-applied biosolids.     

Leaching of phage from Class B biosolids and potential transport through soil

The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.     

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli

Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by real-time polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5–1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.     

Resistance of Faecal Coliforms and Enterococci Populations in Sludge and Biosolids to Different Hygienisation Treatments

The composition of the most abundant facultative anaerobic bacteria populations [faecal coliforms (FC) and enterococci (ENT)] in sludge can be modified after different treatments. These involve the disposal or reuse of sludge and include: anaerobic digesters, incineration, composting, pasteurization and lime treatments. In this study, three treatment types (mesophilic anaerobic digestion, composting and pasteurization) were compared in terms of their ability to reduce both bacterial populations. The diversity and any changes in composition of main phenotypic groups for both populations were also analyzed. Mesophilic anaerobic digestion (MAD) was carried out at 35°C for 20 days. Digested sludge was then dehydrated by centrifugation at 2,500 rpm. Composting (COM) was performed at 55°C with windrow phases. Pasteurization was assayed at 60°C for 90 min (P60), at 80°C for 60 min (P80). A 1–1.5 log unit reduction was observed for FC, and 1 log unit reduction was noted for ENT by MAD treatment. In composting, this reduction proved higher for FC than for ENT (6 log and 3–4 log units, respectively). Optimal pasteurization was obtained at 80°C for 60 min, resulting in a 5 log unit reduction for FC and a 2 log unit reduction for ENT. High diversity indices (Di) for both bacterial populations were detected both before and after implementation of the different treatments. Analyses of the population’s similarity provided that FC were diverse both before and after COM, P60 and P80 treatments. However, no differences were observed on the composition of ENT populations after the different treatments assayed.     

Nutritional status of different biosolids and their impact on various growth parameters of wheat (Triticum aestivum L.)

Biosolids can be effectively recycled and applied as soil amendments for agricultural crops because they contain several important micro and macronutrients including nitrogen, phosphorus, potassium, manganese. In the current study, we evaluated the effectiveness of seven biosoilds on different growth parameters of wheat crop. The biosolids used were lime stabilized, composted, liquid mesophilic anaerobically digested (liquid MAD), thermally dried mesophilic anaerobically digested (thermally dried MAD), thermally hydrolyzed mesophilic anaerobically digested (thermally hydrolysed MAD), dewatered mesophilic anaerobically digested (dewatered MAD) and thermally dried raw biosolids. We also analysed biosolids for their nutrient contents before application. The results revealed that different types of biosolids differed in nitrogen and phosphorous contents with highest contents observed in dewatered (5.70% nitrogen, 2.32% phosphorous) and liquid biosolids (2.35% phosphorous). The plant height, plant diameter and dry weight yield of wheat was increased with the increase in concentrations of biosolids. Liquid MAD resulted in maximum plant height of 120.35 ± 3.23, 133.2 ± 3.67 and 147.25 ± 3.11 at 3.33, 6.66 and 9.99 tons/ha concentration. The highest plant diameter was recorded (1.05–1.45 cm) where mineral nitrogen was applied. The study will be helpful in replacing the synthetic fertilizer with biosolids to fulfil the nutritional requirements of agricultural crops.     

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.     

Feedstocks Affect the Diversity and Distribution of Propionate CoA-Transferase Genes (pct) in Anaerobic Digesters

Anaerobic digestion (AD) is an attractive microbiological technology for both waste treatment and energy production. Syntrophic acetogenic bacteria are an important guild because they are essential for maintaining efficient and stable AD operation. However, this guild is poorly understood due to difficulties to culture them. In this study, we developed specific PCR assays targeting the propionate-CoA transferase genes (pct) to investigate their diversity and distribution in several mesophilic anaerobic digesters and a bench-scale temperature-phased AD (TPAD) system. Phylogenetic analysis of sequenced pct amplicons revealed the occurrence of Syntrophobacter fumaroxidans and six other clusters of putative pct genes. Principal coordinate analysis (PCoA) showed that pct diversity and abundance were largely correlated to the feedstocks of the digesters, while little difference was seen between the granular and the liquid fractions of each digester or between the two digesters of the TPAD system. Cluster-specific qPCR analysis revealed major impact of feedstocks and fractions on the abundance of pct genes. Readily fermentable substrates such as sugar- or starch-rich feedstocks selected for pct genes (Cluster I) related to Syntrophobacter, while manure feedstock selected for pct clusters related to pct of Clostridium spp. These results suggest that propionate metabolism can be affected by feedstocks and partition differently between solid and liquid phases in digesters. The PCR assays developed in this study may serve as a tool to investigate propionate-oxidizing bacteria in anaerobic digesters and other anaerobic environments.     

Dynamics of carbon,nitrogen and phosphorus in soil amended with irradiated,pasteurized and limed biosolids

Sewage biosolids contain high concentrations of pathogens, which limits their use as soil amendment. This study investigated how application of lime (Ca(OH)2), irradiation, or pasteurization reduced pathogens in biosolids and how its application affected soil characteristics. A soil sampled outside the canopy of Mesquite trees (Prosopis laevigata) and from a pasture at Lerma (Mexico) was amended with treated or untreated biosolids, characterized and incubated aerobically while dynamics of carbon (C), nitrogen (N) and phosphorus (P) were monitored. Heavy metals concentrations in the biosolids were low, so it was of excellent quality (USEPA). The amount of pathogens in the biosolids made it a class "B" (USEPA) which can be used in forests. Only irradiation sufficiently reduced faecal coliforms to make it a class "A" biosolids without restrictions in application. C mineralization increased significantly when biosolids were added, but not concentrations of available P (P < 0.05). Ammonium (NH4+) concentrations in soil amended with biosolids were higher compared to unamended soil, but not the concentrations of nitrate (NO3-) except when biosolids treated with Ca(OH)2 was added to the Lerma soil.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Influence of long‐term land application of Class B biosolids on soil bacterial diversity

Aim: To evaluate the effect of long‐term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. Methods and Results: Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0–30 cm) were collected from control (unamended) and biosolid‐amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI Blast N and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids‐amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. Conclusion: Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. Significance and Impact of the Study: This study illustrates that long‐term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity.     

Quantification of Salmonella spp. in composted biosolids using a TaqMan qPCR assay

Composting is increasingly used to transform biosolids, obtained following wastewater treatment, into a more stable organic product that can be released in the environment. The process must however be closely monitored to assure that the end product meets the regulations set by environmental agencies with regards to the amount of pathogenic microorganisms present. In this study, a TaqMan qPCR approach targeting the invA gene was developed to monitor the presence of Salmonella spp. in composted biosolids. A validation step was first performed to evaluate the effect of compost age on the quantification of various concentrations of seeded Salmonella typhimurium. Secondly, qPCR was used to investigate the effect of composting time, varying from 1 month to 24 months, on the presence of Salmonella spp. naturally present in biosolids samples. Culture media were used in parallel to corroborate the results obtained by qPCR. The detection limit of the invA gene obtained experimentally from composts seeded with S. typhimurium was 5.8 copies or the equivalent of 5.8 CFU per qPCR reaction. Although the results indicated that compost age had a marginal effect on the detection of seeded S. typhimurium, the TaqMan qPCR approach was efficient at detecting and quantifying the amount of Salmonella spp. present in naturally contaminated composted biosolids of different ages. Results showed that there was a significant decrease in the amount of Salmonella DNA present in composted biosolids over time, which was also corroborated by the CFU counts obtained on the BSA culture medium. However, qPCR was more specific, robust and rapid to execute than performing counts on culture media. qPCR shows promise for routine examination of composted biosolids to ascertain that pathogenic microorganisms, including Salmonella spp., are decreased below acceptable limits before their application in the environment.